Optimization of L-asparaginase production from Escherichia coli using response surface methodology

Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. In previous study, the L-asparaginase from Erwinia chrysanthermy was expressed in Escherichia coli BL21(DE3). The recombinant L-asparaginase was produced from recombinant E.coli BL21(DE3) under different cultivation conditions (inducer concentration, inoculum concentration and KH2PO4 concentration). The optimized conditions by response surface methodology using face centered central composite design. The analysis of variance coupled with larger value of R2 (0.9) showed that the quadratic model used for the prediction was highly significant (p 0.05). Under the optimized conditions, the model produced L-asparaginase activity of 123.74 U/ml at 1.03 mM IPTG, 3% (v/v) inoculum and 0.5% (w/v) KH2PO4. Recombinant protein was purified by two step using gel filtration and DEAE chromatography. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 462 U/mg and identified by MALDI-TOF mass spectrometry. Results of MALDI-TOF analysis confirmed that recombinant protein was L-asparaginase II. Recombinant L-asparaginase has antiproliferative activity with K562 cell line. In conclusion, this study has innovatively developed cultivation conditions for better production of recombinant L-asparaginase in shake flask culture

Expression, purification and evaluation of recombinant L-asparaginase inmehthylotrophic yeast Pichia pastoris: Expression, purification and evaluation of recombinant L-asparaginase in mehthylotrophic yeast Pichia pastoris: Research article

L-asparaginase (EC 3.5.1.1), a therapeutic enzyme used in the treatment of childhood acute lymphoblastic leukemia (ALL). Hence, the goal of this work is study the expression and evaluation of hydrolysis activity of native sequence (X12746) encoding for L-asparaginase from Erwinia chrysanthemi NCPBB1125 in the popular expression system Pichia pastoris. The sequence of asn encoded for mature protein was expressed in P. pastoris SMD1168 and X33. SDS-PAGE analysis showed recombinant L-asparaginase was secreted efficiently. Stable and high hydrolysis activity of extracellular L-asparaginase in P. pastoris SMD1168 making it a potential candidate to produce recombinant protein. After purification, a specific band whose appearance approximately 45 kDa indicating the glycosylated protein with specific activity by 6.251 Umg-1 and about 3 folds purifications.L-asparaginase (EC 3.5.1.1), một loại enzyme được sử dụng trong điều trị bệng ung thư bạch cầu mãn tính ở trẻ em. Mục tiêu của nghiên cứu này là biểu hiện và đánh giá hoạt tính thủy phân của L-asparaginase mã hóa bởi đoạn gene (X12746) tương ứng từ Erwinia chrysanthemi NCPBB1125 được biểu hiện trong nấm men Pichia pastoris. Gene đã được cắt signal peptide và biểu hiện trong P. pastoris SMD1168 and X33. Qua phân tích kết quả điện di SDS-PAGE của môi trường sau lên men, L-asparaginase tái tổ hợp được tìm thấy trong dịch ngoại bào của P. pastoris. Với khả năng sản xuất protein có hoạt tính cao hơn so với chủng P. pastoris X33, SMD1168 được lựa chọn để biểu hiện L-asparaginase tái tổ hợp. Sau khi tinh sạch, sự xuất hiện của một băng có kích khối lượng phân tử xấp xỉ 45 kDa trên điện di SDS-PAGE cho thấy protein tái tổ hợp đã bị glycosyl hóa với hoạt tính riêng 6.251 Umg-1 và đạt độ sạch 3.471 lần

Selection and identification of thermophilic yeast strains in leachate from the organic waste heap in Phu Luong district, Thai Nguyen province, Vietnam

The study was carried out to isolate and select useful thermophilic yeast strains in the process of organic domestic waste treatment in Phu Luong - Thai Nguyen. Research results from 23 samples of rust have isolated 10 strains of yeast on YPG medium at 40 oC. Among them, 6 strains of yeast were selected with the ability to grow and develop in a wide temperature range from 20-45 oC. The results of identification combined with morphological, physiological, and biochemical characteristics of yeast strains showed that, out of 6 selected strains, there were 3 strains belonging to the genus Saccharomyces (Saccharomyces sp. TNY13.01, Saccharomyces sp. TNY22.01), Saccharomyces cerevisiae TNY13.09), 2 strains of the genus Candida (Candida sp. TNY23.01, Candida tropicalis TNY23.126) and 1 strain of the genus Papiliotrema (Papiliotrema laurentii TNY23.127). Among them, the identified strain Saccharomyces cerevisiae TNY13.09 has the ability to grow at 45, tolerates a wide pH range of 4.0– 8.5, has a positive catalase reaction, is capable of using a variety of carbon sources, and belongs to class I biosafety group. On that basis, Saccharomyces cerevisiae TNY13.09 has the potential to be further researched and applied as additional microbial inoculants to the organic waste heap

Phrasal semantic distance for vietnamese textual document retrieval

In this paper, a computational semantic method is proposed to estimate the phrasal semantic distance used in our model of a Vietnamese document retrieval system. The semantic distances between phrases are defined in terms of semantic classes and semantic relations to ensure that it can reflect how different two certain phrases are. To estimate the semantic distance, the semantic classes of a phase are identified by using the n-gram model. After identification of the semantic classes, their semantic relations are also identified by using a Vietnamese Lexicon Ontology. This handcrafted ontology contains defined semantic classes and their potential relations in Vietnamese language explicitly. For the evaluation purpose, a phrasal semantic retrieval system has been built to test with a data set of 720 phrases and 30 queries. The evaluation shows the precision of 96.6% and the recall of 78.4% on experimental results

IDENTIFICATION AND CHARACTERIZATION OF A PURPLE NONSULFUR BACTERIUM ISOLATED FROM COASTAL AREA OF HAI PHONG FOR USING IN PRODUCTION OF UNSATURATED FATTY ACID (OMEGA 6, 7, 9)

Purple nonsulfur bacteria are a group that has so much biotechnological applications, particularly in producing of functional food rich with unsaturated fatty acids. A purple nonsulfur bacterium (named HPB.6) was chosen based on its strong growth, high lipid and synthesis of unsaturated fatty acid (omega 6,7,9). Studying on basic biological characteristics showed that the cells of HPB.6 were observed as ovoid-rod shape, none motility, Gram negative staining. The diameter of single bacterium was about 0.8-1.0 µm. The cells divide by binary fission and had bacteriochlorophyll a (Bchl a). This bacterium grew well on medium with carbon and nitrogen sources such as acetate, succinate, pyruvate, butyrate, glutamate, arginine, leucine, tyrosine, alanine, methionine, threonine, glutamine, yeast extract and NH4Cl. This selected strain grew well on medium with salt concentrations from 1.5 - 6.0% (optimum 3%), pH from 5.0 to 8.0 (optimum at pH 6.5) and could withstand Na2S at 4.0 - 5.2 mM. Based on morphological, physiological properties and 16S rRNA analysis received demonstrated that HPB.6 strain belongs to the species Rhodovulum sulfidophilum

Cloning and expression of pigC gene in Escherichia coli

Prodigiosin (Pg), which is particularly of interest because of anticancer and antimicrobial activities, can be produced through the PigC-catalyzed condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Therefore, the PigC protein plays an important role in prodigiosin biosynthetic pathway. However, studies related to PigC protein have not been carried out in Vietnam yet. In this work, the pigC gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Using PCR and universal primers, we amplified a fragment of 3 kb covering entire coding region of the pigC gene from Serratia sp. strain M5. The pigC gene was inserted into pJET1.2 vector, and then transformed into E. coli DH10B. The sequence of a recombinant vector pJET1.2/pigC was evaluated by using whole colony PCR amplification. Sequence alignment results revealed that the obtained pigC gene possesses 71.5% and 75.4% of nucleotide identity in comparison with two strains, Serratia 39006 and Serratia sp. AS9 published in GenBank with their respective accession numbers of AJ833001 and CP002773. The recombinant vector pJET1.2/pigC was used to reamplify pigC, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing recombinant vector pET22b/pigC was expressed in the auto-induced medium. The presence of PigC protein in the lysate was identified as a 100 kDa band through Western Blot analysis using anti his-tag antibody. Afterward, the PigC protein was purified by Ni-NTA column, and its expression level was quantified through SDS-PAGE analysis. The results of our study provide a potential material for producing prodigiosin from recombinant protein in Vietnam

Effects of imidacloprid and fenobucarb on the dynamics of the psyllid Diaphorina citri Kuwayama and on the incidence of Candidatus Liberibacter asiaticus

Introduction. The effects of imidacloprid and fenobucarb insecticides on the dynamics of the psyllid Diaphorina citri and on the incidence of Candidatus Liberibacter asiaticus ( Ca. L. a.), the putative causal agent of Huanglongbing disease (HLB), were studied in a field experiment. Materials and methods. The experimental design consisted of three independent 0.5-ha Citrus orchards planted with disease-free HLB-susceptible orange trees, located in a Citrus producing area seriously affected by HLB. Imidacloprid was applied monthly to the trunks in one orchard at a rate of 0.15 g a.i.·tree –1 ; fenobucarb was sprayed fortnightly in a second orchard at a rate of 250 g a.i.·ha –1. The 3rd orchard was managed as a control without insecticide applications. The total number of adult D. citri specimens and the percentages of trees harbouring psyllid eggs and 5th instar nymphs were monitored at fortnightly intervals in each orchard. Ca. L. a. incidence was assessed in each orchard by PCR at 5 months, 12 months and 24 months after planting. Results. Compared with the control, both the fenobucarb and imidacloprid treatments reduced adult psyllid populations by over 90% and reduced the frequency of trees harbouring eggs and 5th instar nymphs. Only imidacloprid treatments totally prevented development of a new generation of adults from eggs. Two years after planting, the prevalence of Ca. L. a. was 0.939, 0.745 and 0.239 in the control and in the orchards treated with fenobucarb and imidacloprid, respectively. Discussion and conclusion. The results indicated that, although both the insecticides used effectively reduced D.citri populations by killing adults and nymphs and by affecting or preventing psyllid reproduction in orchards, neither of the two insecticide treatments totally prevented transmission of Ca. L. asiaticus. However, due to its long-lasting effect and systemic activity, the imidacloprid treatment provided the best protection against infections, and delayed and slowed down the spread of the pathogen. Furthermore, it reduced the number of pesticide applications needed and left the way open for biological integrated pest management programmes. (Résumé d'auteur

Mutagenesis development of actinoplanes sp. KCTC 9161 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and screening for acarbose production

Acarbose has been widely used in the therapy of type II diabetes (non-insulin dependent) because it controls blood sugar contents of patients after meals. Acarbose, a pseudo-oligosaccharide, acts as a competitive -glucosidase inhibitor. Acarbose is produced by the strains of Bacillus, Streptomyces and Actinoplanes sp. The aim of this study was to develop mutagenesis for an Actinoplanes sp. strain and screening for acarbose production. The spores of Actinoplanes sp. KCTC 9161 strain were subjected to be mutated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) for screening and finding mutant strains that were capable of production of higher acarbose (an inhibitor of α-glucosidase) higher than wild type strain. Firstly, the original NTG solution was prepared in phosphate buffer 0.05 M, pH 6.9 and the safety concentration of NTG was determined at 5 mg/ml. Then, the spores were incubated with different NTG amounts and duration. The living colonies were transferred to fermentation medium. The results obtained showed that 15 mutant strains were produced higher acarbose than wild type when used thin layer chromatography method for analysis and comparing with standard acarbose (Sigma). Three cell lines among total tested 15 mutant lines of Actinoplanes sp. KCTC 9161 produced acarbose at a higher level or indicated a higher inhibitory activity toward α-glucosidase than the original strain. Enzymatic inhibitory ativity of α-glucosidase of three mutant strains (Actinoplanes sp. KCTC- L4, L11, L14) was increased 1.3 fold higher than wild type and Actinoplanes sp. KCTC spores were very sensitive to NTG toxic, 98% spores could not survive at the treatment condition of 50 µg NTG for 30 minutes. In addition, an applicable protocol for mutating Actinoplanes sp. using NTG was suggested for further research

SỰ BIẾN ĐỔI CỦA MỘT SỐ THÀNH PHẦN POLYPHENOL Ở VỎ QUẢ VẢI THIỀU THANH HÀ SAU THU HOẠCH

The colour change of pericarp of postharvest litchi fruits is related to the formation of melannin, that is caused by enzymes involving in catalysis of melanin biosynthesis pathway in the litchi pericarp, in this respect polyphenol oxidase (PPO) enzyme is the most important one. This enzyme is involved in the first and the second reactions of the melanin biosynthesis reaction chain. PPO substrates are polyphenol compounds which can be found in litchi pericarp as anthocyanins and flavonoids. The results of our experiments showed that the concentration of melanin in lychee pericarp in 180 hours after being harvested increased by 7.169 times at the room temperature and by 5.05 times at 8oC. Total polyphenol content after 180 hours at room temperature and at 8oC remains 19.2% and 17.66%, respectively. The anthocyanin content after 180 hours almost completely lost at room temperature and remains 69.03% at 8oC. Total flavonoid content after 180 hours at room temperature and at 8oC remains 31.78% and 46.92%, respectively.Hiện tượng nâu hóa sau thu hoạch ở vỏ quả vải thiều (Litchi Chinensis Sonn.) liên quan đến sự hình thành của melannin trong vỏ. Phản ứng sinh tổng hợp melanin là một chuỗi phản ứng với sự tham gia của một số enzyme nhưng enzyme polyphenol oxydase (PPO) là enzyme quan trọng nhất, enzyme này tham gia vào phản ứng đầu tiên và phản ứng thứ hai của chuỗi phản ứng. Cơ chất của PPO là các hợp chất polyphenol có nhiều trong vỏ quả vải, như anthocyanin và flavonoid. Quá trình biến nâu làm biến đổi hàm lượng của melanin và các thành phần polyphenol ở vỏ quả vải thiều. Kết quả thí nghiệm của chúng tôi cho thấy, nồng độ của melanin ở vỏ quả vải thiều Thanh Hà (VTTH) sau khi thu hoạch 180 giờ tăng gấp 7,169 lần ở nhiệt độ phòng và tăng gấp 5,05 lần ở 80C. Hàm lượng polyphenol tổng số sau 180 giờ ở nhiệt độ phòng còn lại 19,2% và 17,66% ở 80C. Hàm lượng anthocyanin sau 180 giờ gần như mất hoàn toàn ở nhiệt độ phòng và còn lại 69,03% ở 80C. Hàm lượng flavonoid tổng số sau 180 giờ còn lại 31,78% ở nhiệt độ phòng và 46,92% ở  80C